The major economic and food value of most agricultural plant products resides in their seeds, and seeds have long been the major resources of proteins, carbohydrates, and oils. Centuries of agricultural research have been directed to improving the qualitative and quantitative traits associated with seed products; classical breeding techniques have resulted in the development of new varieties with desirable traits not observed in the source populations from which the new varieties are developed. However, despite recent rapid progress, these techniques are limited to recombining genetic information which is already present in the source population, and to the very slow modification of this information by naturally occurring mutations. Furthermore, these classical methods may also result in undesirable traits arising as a consequence of selecting for a particular desirable trait. For example, it was impossible to increase the oleic acid content of rapeseed oil above 80% without obtaining undesired agronomic properties such as reduced cold tolerance; it was hypothesized that the observed reduction in cold tolerance was due to the lack of unsatured fatty acids in the membranes of these plants (Kinney, Current Opinion in Biotechnology, 5:144-151 (1994); Miquel et al., Plant Physiology, 106:421-427 (1994)). Thus, the characteristic of high oleic acid, which is desirable when present in the seed oil (which consists primarily of storage lipids, or triacylglycerols), is undesirable when present in the membrane lipids (which consist primarily of glycerolipids).
The application of the newer techniques of genetic engineering promises to revolutionize plant agriculture. It is envisioned that traditional seed products can be tailored to the end market, as for example, seed oils produced with specific fatty acid profiles. Thus, it has been possible to produce a rapeseed line with 88% oleic acid in the triacylglycerol fraction of the seed oil, by transferring an antisense gene to a fatty acid desaturase, FAD2, to the rapeseed; this desirable characteristic was limited to the seed oils, and therefore did not affect the fatty acids of the membrane lipids of the rest of the plant, by putting the antisense gene under control of the napin seed-specific promoter (Hitz et al., Kader, J.-C. and Mazliak, P., Eds. (Kluwer, Dordecht, Netherlands), p. 534 (1995)). It is also envisioned that seeds can be used produce non-traditional products, such as edible vaccines. However, for these applications as well, it is preferable to utilize seed specific promoters, to limit the presence of such non-traditional products to the seed, and to avoid their presence in other parts of the plant.
Only a few seed-specific promoters have been cloned and studied in detail; these include promoters for seed storage protein genes, such as a phaseolin promoter (U.S. Pat. No. 5,504,200) and a napin promoter (U.S. Pat. No. 5,608,152). Storage proteins are usually present in large amounts, making it relatively easy to isolate storage protein genes and the gene promoters. Even so, the number of available seed specific promoters is still limited. Furthermore, most of these promoters suffer from several drawbacks; they have a limited period of time during seed development in which they are active, and they may be expressed in other tissues as well. For example, storage protein gene promoters are expressed mainly in the mid to late embryo development stage (Chen et al., Dev. Genet., 10(2): 112-122 (1989); Keddie et al., Plant Mol. Biol., 19(3):443-53 (1992); Sjodahl et al., Planta., 197(2):264-71 (1995); Reidt et al., Plant J., 21(5):401-8 (2000)), and also may have activity in other tissues, such as pollen, stamen and/or anthers (as, for example, the phaseolin promoter, as reported by Ahm, V, et al. Plant Phys 109:1151-1158 (1995)).
Therefore, it would be desirable to have additional seed-specific promoters for use in modifying seed products. It would also be desirable to have seed-specific promoters which are more tightly expressed only in seed tissue. It would also be desirable to have seed-specific promoters which are active during different phases of seed development, and which are active to different degrees during seed development It would also be desirable to have a method by which such seed-specific promoters can be identified.